There has been much effort to clone restriction-modification systems. The first cloning of a DNA endonuclease gene was described by Mann MB et al. (1978) Gene 3:97-112. Since then more than seventy DNA methylase and restriction endonucleases have been cloned, the majority of the restriction endonuclease genes being closely linked to its corresponding methylase gene. Cloning of such genes allows one to produce large quantities of an enzyme.
Several methods by which restriction-modification systems can be cloned have been described. A number of endonuclease and methylase genes have been cloned from endogenous plasmids: EcoRII (Kosykh V. B. et al. (1980) Mol. Gert. Genet. 178:717-718), EcoRI (Newman A. K. et al. (1981) J. Biol. Chem. 256:2131-2139, Greene P. J. et al. (1981) J. Biol. Chem. 256:2143-2153), EcoRV (Bougueleret L. et al. (1984) Nucl. Adds Res. 12:3659-3676), PvuII (Blumenthal R. M. et al. (1985) J. Bacteriol. 164:501-509), and PaeR71 (Gingeras T. R. and Brooks J. E. (1983) Proc. Natl. Acad. Sci. USA 80:402-406). Other methods of cloning include a phage restriction method in which bacterial cells carrying cloned restriction and modification genes will survive phage infection (Mann et al. supra; Walder R. Y. et al. (1981) Proc. Natl. Acad. Sci. U.S.A. 78:1503-1507; Rodicio M. R. and Chater K. F. (1988) Mol. Gen. Genet. 213:346-353), and a procedure based on methylation protection suggested by Mann et al., supra, and Szomolanyi E. et al. (1980) Gene 10:219-225. This latter scheme involves digestion of a plasmid library with the restriction enzyme to be cloned so that only plasmids whose sequences are modified, because of the presence of the methylase, will produce transformants in a suitable host. This selection has worked well to clone endonuclease and methylase genes together as well as methylase genes alone (Szomolanyi et al., supra; Janulaitis A. et al. (1982) Gene 20:197-204; Walder R. Y. et al. (1983) J. Biol. Chem. 258:1235-1241; Kiss A. and Baldanf F. (1983) Gene 21:111-119; Wilson G. G. (1988) Gene 74:281-289). However, this technique sometimes yields only the methylase gene, even though the endonuclease and modifying genes are closely linked.
Cloning of certain restriction-modification systems in E. coli, including DdeI (Howard K. A. et al. (1989) Nucl. Acids Res. 14:7939-7950), BamHI. (Brooks J. E. et al. (1989) Nucl. Acids Res. 17:979-997), KpnI (Hammond AW et al. (1990) Gene 97:97-102), ClaI (U.S. application Ser. No. 08/002,032) and NsiI (disclosed herein), has required a multi-step approach. In each case, protection of the host with methylase expressed on a plasmid was necessary to stabilize a compatible vector containing the functional endonuclease gene. It would appear that in these cases, simultaneous introduction of the methylase and endonuclease genes results in degradation of the cell genome because the endonuclease is able to cleave the unmodified sites before the methylase is able to protect them. If the methylase is already in the cell at the time the endonuclease gene is introduced, there should be no unmodified sites and the endonuclease gene is not toxic.